Identification of Stably Expressed lncRNAs as Valid Endogenous Controls for Profiling of Human Glioma

Abstract

Background: Recent research indicates that long non-coding RNAs (lncRNA) represent a new family of RNAs that is of fundamental importance for controlling transcription and translation. Thereby, there is increasing evidence that lncRNAs are also important in tumourigenesis. Thereby valid expression profiling using quantitative PCR requires suitable, stably expressed normalisers to achieve reliable and reproducible data. However, no systematic analysis of suitable references in lncRNA studies in human glioma has been performed yet.

Methods: In this study, we investigated 90 lncRNAs in 30 tissue specimen for the expression stability in human diffuse astrocytoma (WHO-Grade II), anaplastic astrocytoma (WHO-Grade III) and glioblastoma (WHO-Grade IV) both alone as well as in comparison with normal white matter. Our identification procedure included a rigorous bioinformatical selection process that resulted in the inclusion of only highly abundant, equally expressed lncRNAs for further analysis. Additionally, lncRNAs were classified according to their stability value using the NormFinder algorithm.

Results: We identified 24 appropriate normalisers suitable for studies in diffuse astrocytoma, 22 for studies in anaplastic astrocytoma and 12 for studies in glioblastoma. Comparing all three glioma entities 7 lncRNAs showed stable expression levels. Addition of normal brain tissue resulted in only 4 suitable lncRNAs.

Conclusions: Our findings indicate that 4 lncRNAs (HOXA6as, H19 upstream conserved 1 and 2, Zfhx2as and BC200) are suitable as normalisers in glioma and normal brain. These lncRNAs may thus be regarded as universal references being applicable for the accurate normalisation of lncRNA expression profiling in various glioma (WHO-Grades II-IV) alone and in combination with brain tissue. This enables to perform valid longitudinal studies, e.g. of glioma before and after malignisation to identify changes of lncRNA expressions probably driving malignant transformation.

Keywords: Glioma; Profiling.; References; lncRNA; long non-coding RNA; qPCR.

Figure 1

Bi-hierarchical clustering of all 90 investigated lncRNAs in 30 tissue specimen. We investigated 5 diffuse astrocytomas (A II, DA, WHO-Grade II), 5 anaplastic astrocytomas (A III, AA, WHO-Grade III), and 15 glioblastomas (GBM, WHO-Grade IV) as well as 5 occipital white matter tissues (WM) as healthy control. There is a clear clustering of tumour entities visible. Blue: low Ct-value; red: high Ct-value.

Figure 2

Identification of stable lncRNAs in diffuse astrocytoma in different glioma entities. In diffuse astrocytoma (WHO-Grade II) we identified 24 (A), in anaplastic astrocytoma (WHO-grade III) we identified 22 (B) and in glioblastoma (WHO-Grade IV) we identified 12 lncRNAs suitable as normalisers (C). Indicated are mean and SD.

Figure 3

Stably expressed lncRNAs suitable for studies comparing different glioma entities. We identified 7 lncRNAs that are suitable as normalisers in studies investigating different glioma entities. Indicated are mean and SD.

Figure 4

Identification of lncRNAs suitable as normalisers in studies investigating glioma and normal brain tissue. We identified 4 lncRNAs that show stable expression in different glioma entities and in normal white matter. These normalisers can be regarded as universal normalisers that are suitable as references in a broad range of lncRNA studies. Indicated are mean and SD.